RESUMEN
All analytical techniques have experienced major progress since the last ten years and electroanalysis is also involved in this trend. The unique characteristics of phenomena occurring at the electrode-solution interface along with the variety of electrochemical methods currently available allow for a broad spectrum of applications. Potentiometric, conductometric, voltammetric and amperometric methods are briefly reviewed with a critical view in terms of performance of the developed instrumentation with special emphasis on pharmaceutical and biomedical applications.
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Técnicas de Química Analítica , Técnicas Electroquímicas , Investigación Biomédica , Electroquímica , FarmaciaRESUMEN
A silver amperometric detector coupled to liquid chromatography (LC) was used for the determination of 6-thioguanine (6-TG) and two of its metabolites, thiouric acid (TU) and 2-amino-6-mercaptopurine riboside (6-TGR). The silver detector coupled to LC operated at a low applied potential (0.08 V vs Ag/AgCl) and offered a chromatogram with peak responses corresponding to molecules interacting with silver, namely, chloride ions and small soluble biothiols in addition to the organothiol drug compounds investigated. Online electrochemical surface cleaning permitted the improvement of the repeatability and peak shape of the recorded signal compared to direct current amperometric detection (AD) when operating in chloride containing media. The studied molecules were eluted isocratically within 5 min on a reversed-phase C18 column without interference from endogenous biothiols present in urine samples. Diluted urine samples (1:1) were directly injected in the LC setup; a linear calibration curve was obtained between peak area and analyte concentration between 0.1 and 10 µM for all the studied molecules. Limits of detection (LODs) were 0.03, 0.008, and 0.01 µM, and the limits of quantification (LOQs) were 0.1, 0.02, and 0.03 µM for TU, 6-TG, and 6-TGR, respectively. Within-day RSDs were 2%, 0.8%, and 1% and between-day RSDs were 2%, 0.9%, 2% for TU, 6-TG, and 6-TGR, respectively. Recoveries in spiked urine were 99.8%, 99.9%, and 99.0% for TU, 6-TG, and 6-TGR, respectively.
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Cromatografía Liquida/métodos , Técnicas Electroquímicas/métodos , Purinas/orina , Plata/química , Compuestos de Sulfhidrilo/análisisRESUMEN
A commercially available thin-layer flow-through amperometric detector, with the sensing block customized in an original design, was applied to the screening of drug compounds known as acetylcholinesterase (AChE) inhibitors. AChE from electric eel was covalently immobilized onto a cysteamine modified gold disk adjacent to a silver disk working electrode. On-line studies were performed by flow injection analysis (FIA) in PBS buffer pH 7.4. Seven commercially available AChE inhibitors used in the medical field, namely neostigmine, eserine, tacrine, donepezil, rivastigmine, pyridostigmine and galantamine as well as two natural compounds, quercetin and berberine, were investigated. The same trend of inhibitory potency as described in the literature was observed. Of particular interest and in addition to the determination of the IC50 values, this flow-through system allowed the study of both, the stability of the enzyme-inhibitor complex and the kinetic of the enzyme activity recovery.
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Inhibidores de la Colinesterasa/análisis , Técnicas Electroquímicas/instrumentación , Enzimas Inmovilizadas/química , Análisis de Inyección de Flujo/métodos , Electrodos , Concentración de Iones de Hidrógeno , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Seeds and aerial parts of Peganum harmala L. are widely used in Algeria as anti-inflammatory remedies. Evaluation of Peganum harmala total alkaloids extracts and pure ß-carboline compounds as an anti-inflammatory treatment by the inhibition of an enzyme key of inflammatory, myeloperoxidase (MPO) and HPLC quantification of the alkaloids from the different parts of plant. MATERIALS AND METHODS: MPO inhibition was tested using taurine chloramine test. The inhibition of LDL oxidation induced by MPO was carried out. The molecular docking analysis of Peganum harmala alkaloids on MPO was performed using the Glide XP docking protocol and scoring function and the redox potential of alkaloids was determined using an Epsilon potentiostat. The concentration of harmala alkaloids was determined using HPLC analysis. RESULTS: The HPLC profiling of the active total alkaloids indicates that ß-carboline e.g. harmine, harmaline, harmane, harmol and harmalol are major components. As ß-carbolines resemble tryptamine, of which derivatives are efficient inhibitors of MPO, the harmala alkaloids were tested for their activity on this enzyme. Total alkaloids of the seeds and of the aerial parts strongly inhibited MPO at 20µg/mL (97±5% and 43±4%, respectively) whereas, at the same concentration, those of the roots showed very low inhibition (15±6%). Harmine, harmaline and harmane demonstrated a significant inhibition of MPO at IC50 of 0.26, 0.08 and 0.72µM respectively. These alkaloids exerted a similar inhibition effects on MPO-induced LDL oxidation. Molecular docking analysis of Peganum harmala alkaloids on MPO showed that all active Peganum harmala alkaloids have a high affinity on the active site of MPO (predicted free energies of binding up to -3.1kcal/mol). Measurement of redox potentials versus the normal hydrogen electrode clearly differentiated (i) the high MPO inhibitory activity of harmine, harmaline and harmane (+1014, 1014 and 1003mV, respectively); and (ii) the low activity of harmalol and harmol (+629/778 and 532/644mV, respectively). A reverse phase HPLC method has been developed to determine simultaneously five alkaloids of Peganum harmala. Seeds contained all five ß-carboline derivatives with the main active alkaloids, harmaline and harmine, being up to 3.8% and 2.9%, respectively. Up to 3.2% of harmine was determined in the roots. The four ß-carboline derivatives, harmine, harmaline, harmane and harmalol were identified in the aerial parts. The highest inhibitory effect observed in seeds and the moderate effect of aerial parts could be explained by their harmine and harmaline content. In contrast, the very weak inhibition of the root extract, despite the presence of harmine, may tentatively be explained by the high concentration of harmol which can reduce Compound II of MPO to the native form. CONCLUSION: The inhibition of MPO by Peganum harmala ß-carboline alkaloids, herein reported for the first time, may explain the anti-inflammatory effect traditionally attributed to its herbal medicine.
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Alcaloides/farmacología , Inhibidores Enzimáticos/farmacología , Peganum/química , Peroxidasa/antagonistas & inhibidores , Extractos Vegetales/química , Alcaloides/aislamiento & purificación , Sitios de Unión , LDL-Colesterol/química , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/aislamiento & purificación , Etnofarmacología , Humanos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Estructura Molecular , Oxidación-Reducción , Peroxidasa/química , Componentes Aéreos de las Plantas/química , Raíces de Plantas/química , Semillas/químicaRESUMEN
The present work aimed at showing the interest in applying liquid chromatography with amperometric detection at a silver electrode (LC-EC-Ag) in order to record a chromatogram highly selective to non volatile aminothiols present in white wines. By integrating and summing the peak area of the aminothiols and by normalizing with respect to the peak area of an injected standard solution of glutathione (1 µM) a new quantitative criteria for white wine characterization is proposed namely, the glutathione equivalent capacity (GEC). The LC setup uses a C18 column in isocratic mode and the analysis takes less than 4 min. The wine sample needs no sample treatment other than dilution with the mobile phase. This new methodology and concept is illustrated by the LC-EC-Ag analysis of several white wines of different origins especially Alsace Riesling wines and Riesling grape juice. It is anticipated that in addition to the determination of the GEC, the developed method may be of interest for establishing a white wine "signature" based on the chromatographic profile.
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Cromatografía Liquida/métodos , Glutatión/análisis , Vitis/química , Vino/análisis , Cromatografía Liquida/instrumentaciónRESUMEN
Liquid chromatography coupled to a silver electrode based flow-through amperometric detector (LC-EC-Ag) was developed for the determination of aminothiols in white wines. The C18 reversed phase LC system operated in the isocratic mode at 0.7 mL min(-1) and used an acidic mobile phase composed of formic acid, EDTA, sodium nitrate, sodium hydroxide, and methanol 1% (v/v) at pH 4.5. The working electrode operated at 0.08 V vs Ag/AgCl, 3M KCl and its manual cleaning was realized once a month by smoothing on a polishing cloth. The analyzed aminothiols were resolved and eluted within 4 min, and all standard curves were linear in the range 2×10(-7)-2×10(-5) M. The analyzed wine samples needed no preparation other than dilution with the mobile phase. The concentration of cysteine (CYS), homocysteine (HCYS), glutathione (GSH) and N-acetylcysteine (NAC) in bottled white wines, determined by the method of standard addition, was found to be in the low µM range (0.2-2 mg L(-1)) depending on the wine type and its age.
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Aminoácidos Sulfúricos/análisis , Glutatión/análisis , Electrodos de Iones Selectos , Plata/química , Compuestos de Sulfhidrilo/análisis , Vino/análisis , Cromatografía Liquida/métodos , Electroquímica/métodosRESUMEN
Due to its production of potent antimicrobial oxidants including hypochlorous acid, human myeloperoxidase (MPO) plays a critical role in innate immunity and inflammatory diseases. Thus MPO is an attractive target in drug design. (Aminoalkyl)fluoroindole derivatives were detected to be very potent MPO inhibitors; however, they also promote inhibition of the serotonin reuptake transporter (SERT) at the same concentration range. Via structure-based drug design, a new series of MPO inhibitors derived from 3-alkylindole were synthesized and their effects were assessed on MPO-mediated taurine chlorination and low-density lipoprotein oxidation as well as on inhibition of SERT. The fluoroindole compound with three carbons in the side chain and one amide group exhibited a selectivity index of 35 (Ki/IC50) with high inhibition of MPO activity (IC50 = 18 nM), whereas its effect on SERT was in the micromolar range. Structure-function relationships, mechanism of action, and safety of the molecule are discussed.
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Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Indoles/síntesis química , Indoles/farmacología , Peroxidasa/antagonistas & inhibidores , Animales , Ácidos Carboxílicos/química , Cristalización , Diseño de Fármacos , Inhibidores Enzimáticos/toxicidad , Flúor/química , Humanos , Indicadores y Reactivos , Indoles/química , Lipoproteínas LDL/química , Masculino , Modelos Moleculares , Nitrógeno/química , Oxidación-Reducción , Ratas , Ratas Wistar , Inhibidores Selectivos de la Recaptación de Serotonina/síntesis química , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Relación Estructura-Actividad , Azufre/química , Taurina/análogos & derivados , Taurina/farmacologíaRESUMEN
The immobilization of tyrosinase onto glutaraldehyde activated streptavidine magnetic particles and subsequent retention onto a magnetized carbon paste electrode for the amperometric assay of tyrosinase inhibitors is described. Tyrosine was used as substrate as it is the first substrate in the melanogenesis process. The sensing mode is based on monitoring the decrease of the amperometric signal corresponding to the electrochemical reduction of dopaquinone enzymatically generated. This current decrease is due to the presence of inhibitors acting directly on the enzyme or inhibitors acting on the product of the enzymatic reaction, i.e. dopaquinone. The methodology is designed for the evaluation of the inhibitory potency of the most frequently used active substances in cosmetic marketed products against hyperpigmentation such as kojic acid, azelaic acid and benzoic acid. These compounds bind to the tyrosinase active center. Ascorbic acid is also investigated as it interrupts the synthesis pathway of melanin by reducing the melanin intermediate dopaquinone back to l-dopa. By comparing the obtained IC(50), under the same experimental conditions, the order of their inhibitory potency was: kojic acid (IC(50)=3.7 × 10(-6)M, K(i)=8.6 × 10(-7)M), ascorbic acid (IC(50)=1.2 × 10(-5)M), benzoic acid (IC(50)=7.2 × 10(-5)M, K(i)=2.0 × 10(-5)M) and azelaic acid (IC(50)=1.3 × 10(-4)M, K(i)=4.2 × 10(-5)M) in close agreement with literature spectrophotometric inhibition data using the soluble tyrosinase.